The ability of small interfering RNAs and microRNAs to silence the expression of specific genes is among the major biomedical discoveries of the last two decades. However the difficulty of delivering sufficient doses of these labile, hydrophilic macromolecules to disease sites continues to hamper the practical application of RNA interference in medicine. Two key problems are that (i) RNAi therapeutics lack inherent permeability across cellular membrane barriers and (ii) that they are rapidly degraded in the extracellular environment. To address these issues the major existing strategy is to encapsulate RNAi agents within protective nanoparticles that contain cationic components. However, the inherent toxicity of existing delivery vectors have limited the translational capacity of this approach in applications requiring systemic delivery. In contrast, nature has provided an elegant solution for delivering RNAi agents without damaging cellular membranes ? siRNA transport through the cellular gap junction network. Specifically, hexameric connexin channels imbedded in the plasma membranes of adjacent cells come together to form gap junctions, direct passageways between the cytoplasm of a cell and its neighbors, which cells use to share diverse hydrophilic molecules including siRNA. Recently, we have developed novel delivery systems that can harness the potential of the gap junction network. Here we propose to develop RNAi loaded connectosomes, lipid membrane vesicles that incorporate a high concentration of connexin membrane channels, creating a direct passageway from the interior of the vesicle to the cellular interior. Preliminary work demonstrates that using our novel connectosomes to deliver small molecule chemotherapeutics increases the efficiency of tumor cell killing by 1000 fold in comparison to conventional liposomal delivery, results which illustrate the importance of direct access to the cell cytoplasm. Further, we have demonstrated loading of connectsomes with siRNA and the release of siRNA into the cell cytoplasm. These findings, coupled with the well-established role of gap junctions in natural RNAi delivery, suggest our hypothesis ? particles that access the gap junction network can substantially increase the efficiency of siRNA delivery. Two aims will develop siRNA-loaded connectosomes and test their efficacy in both in vitro and in vivo models of breast cancer. Aim 1 is to perform an in vitro optimization of connectosomes for siRNA delivery and gene knockdown. These studies will utilize quantitative measures of siRNA loading, release, and resulting gene knockdown. Then Aim 2 is to perform an in vivo evaluation of the anti-tumor efficacy of siRNA and chemotherapeutics delivered using connectosomes. These studies will utilize a breast tumor xenograft model in mice. Harnessing the gap junction network to circumvent the endosomal pathway for delivery of siRNA and microRNA is highly innovative and has the potential to provide an entirely new route for transport of these potent therapeutics into the cytoplasm. This fundamental conceptual innovation in RNAi delivery will allow us to increase the efficiency and reduce the toxicity of siRNA therapeutics, helping to realize their potential for treatment of diverse diseases.